This invention relates to a process for making and purifying papillomavirus (HPV) virus-like particles (VLPs), which can be used as a vaccine component.
Papillomavirus infections occur in a variety of animals, including humans, sheep, dogs, cats, rabbits, monkeys, snakes and cows. Papillomaviruses infect epithelial cells, generally inducing benign epithelial or fibroepithelial tumors at the site of infection. Papillomaviruses are species specific infective agents; a human papillomavirus cannot infect a nonhuman animal.
Papillomaviruses may be classified into distinct groups based on the host that they infect. Human papillomaviruses (HPV) are further classified into more than 70 types based on DNA sequence homology (for a review, see Papillomaviruses and Human Cancer, H. Pfister (ed.), CRC Press, Inc., 1990). Papillomavirus types appear to be type-specific immunogens in that a neutralizing immunity to infection to one type of papillomavirus does not confer immunity against another type of papillomavirus.
Papillomaviruses are small (50-60 nm), nonenveloped, icosahedral DNA viruses that encode for up to eight early and two late genes. The open reading frames (ORFs) of the virus genomes are designated E1 to E7 and L1 and L2, where xe2x80x9cExe2x80x9d denotes early and xe2x80x9cLxe2x80x9d denotes late. L1 and L2 code for virus capsid proteins. The early (E) genes are associated with functions such as viral replication and cellular transformation.
The L1 protein is the major capsid protein and has a molecular weight of 55-60 kDa. L2 protein is a minor capsid protein which has a predicted molecular weight of 55-60 kDa and an apparent molecular weight of 75-100 kDa as determined by polyacrylamide gel electrophoresis. Immunologic data suggest that most of the L2 protein is internal to the L1 protein. The L2 proteins are highly conserved among different papillomaviruses, especially the 10 basic amino acids at the C-terminus. The L1 ORF is highly conserved among different papillomaviruses.
Recombinant L1 protein has been made in a variety of hosts, and under proper conditions self-assembles into virus-like particles (VLPs), either alone or in combination with L2. VLPs are candidates for a commercial vaccine. However, in order to be useful in a human vaccine, the VLPs must be highly purified and free from host cell contaminants. In the past, cross-flow ultrafiltration in a diafiltration mode has been used to remove contaminating biomolecules. However, this method resulted in the proteolytic degradation of the HPV L1. It would be desirable to have a L1 protein purification process which results in a highly pure, non-degraded product.
This invention relates to a method of purifying recombinant papilloma virus (HPV) virus-like particles (VLPs) comprising the steps of: contacting a partially purified VLP-containing cell lysate with a hydroxyapatite medium in a chromatography column, under conditions such that the VLPs bind to the hydroxyapatite medium; and eluting the bound VLPs with a solution comprising phosphate anions; and recovering the eluted VLPs.
The purification process can be used with VLPs which consist substantially of L1 protein, and it can also be used with VLPs which comprise L1 and L2 proteins. In addition it can be used with VLPs which are chimeric, i.e. contain L1 protein and a L2:fusion protein. In general, for vaccine use, VLPs which contain only L1 proteins are preferred.
The process is applicable to VLPs from virtually any strain of papillomavirus. It is preferred that a human papillomavirus (HPV) be used. Preferred strains of HPV are those which are known to cause the most serious diseases and conditions, including: HPV type 6a, HPV type 6b, HPV type 11, HPV type 16, HPV type 18, HPV type 31, HPV type 33, and HPV type 45.
In general, a host cell is transformed with a vector which encodes L1 or L1 and L2 proteins, or L1 and L2:fusion protein.
As used throughout the specification and claims, the term xe2x80x9cL2: fusion proteinxe2x80x9d means that the DNA encoding the L2 protein has been operatively linked to another DNA encoding a desired protein, and preferably, another protein from HPV such as E1, E2, E3, E4, E5, E6 or E7. The L2 portion of the fusion protein may be full length, or it may have deletions and/or truncations. Examples may be found in co-pending U.S. Provisional Patent Application S. No. 60/096,638, (Attorney Docket Number 20276PV, which is hereby incorporated by reference) filed herewith.
The host cell may be any host cell which is easily cultured, as is known in the art, including yeast (Saccharomyces cerevisiae), insect cells, bacterial or mammalian cells. Yeast cells are particularly preferred.
The vector may also contain other elements as is known in the art, such as transcription and translation controlling elements and/or marker genes. The expressed L1, L1 and L2, or L1 and L2:fusion proteins will spontaneously assemble into VLPs. Host cells are typically lysed, and the cell lysate is then partially purified.
The partial purification step may include commonly used purification steps, and is not seen as a critical step in this invention. For example, the cell lysate may be subjected to a microfiltration process, and to at least one chromatography step, such as a cation-exchange chromatography.
It has been found, in accordance with this invention that a chromatography step, using hydroxyapatite as the column medium, followed by elution with a buffer solution containing phosphate anion, removes a large amount of contaminants from a partially purified cellular lysate. Specifically, it has been found that most contaminating biomolecules, including DNA, lipids and proteins are removed from the lysate.
In accordance with this invention, the final purified VLP preparation is generally at least 75% pure, preferably at least 80% pure, and more preferably at least 90% pure, as measured using the SDS/PAGE assay.
Virtually any commercially available hydroxyapatite column material may be used in this invention. It is preferable to use a ceramic hydroxyapatite which a particle size of approximately 20-50 xcexcm and approximately an 800 xc3x85 pore size. One such commercially available hydroxyapatite is sold by BioRad as xe2x80x9cCeramic hydroxyapatite, Type IIxe2x80x9d. However, others are effective as well.
In preparing the chromatography step of the purification process, it is recommended that the column feed be in a buffer with a pH of 6-8, and preferably approximately 7. A preferred buffer is 50 mM MOPS [3-(N-morpholino)propanesulfonic acid] at a pH of 7.0 and also containing 1.25 M NaCl.
Other buffer systems which may also be used are apparent to one of ordinary skill in the art and include: MES [2-(N-morpholino)ethanesulfonic acid]; BIS-TRIS [bis-(2-hydroxyethyl)-amino]tris-(hydroxymethyl)methane]; ADA [N-2-acetamidoiminodiacetic acid, monosodium salt]; ACES [N-2-acetamido-2-aminoethanesulfonic acid]; PIPES [piperazine-N,Nxe2x80x2-bis(2-ethane-sulfonic acid)]; MOPSO [(3-N-morpholino)-2-hydroxypropane-sulfonic acid]; BIS-TRIS PROPANE [1,3-bis [tris(hydroxymethyl)methyl-amino]propane]; BES [N,N-bis-(2-hydroxyethyl)-2-amino-ethanesulfonic acid]; TES [N-tris(hydroxymethyl)methyl-2-aminoethane-sulfonic acid and 2-2([2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethane sulfonic acid]; HEPES [N-2-hydroxyethylpiperazine-Nxe2x80x2-2-ethane-sulfonic acid]; DIPSO [3-(N,N-bis(2-hydroxyethyl)amino)-2-hydroxy-propanesulfonic acid]; TAPSO [3-N-tris(Hydroxymethyl)methylamino]-2-hydroxy-propanesulfonic acid]; TRIS [tris-(hydroxymethyl)-aminomethane]; HEPPSO [N-(2-hydroxyethyl)-piperazine-Nxe2x80x2-[2-hydroxy-propanesulfonic acid)]; POPSO [(piperazine-N,Nxe2x80x2-bis[2-hydroxypropanesulfonic acid)]; EPPS [N-[2-Hydroxyethyl]-piperazine-Nxe2x80x2-[3-propanesulfonic acid and HEPPS]; TEA [triethanolamine]; TRICINE [N[tris-(hydroxymethyl)methyl]glycine]; BICINE [N,N-bis-(2-hydroxyethyl]-glycine]; TAPS [3-{[tris-(hydroxymethyl)methyl]amino}-propane sulfonic acid]; imidazole; HEPPS [N-2-hydroxyethylpiperazine-Nxe2x80x2-3-propane-sulfonic acid]; glycine amide, hydrochloride; glycylglycine; citrate; acetate; and succinate buffers.
The partially purified VLPs in the buffered solution is allowed to come into contact with the hydroxyapatite medium under conditions which allow the VLPs to bind to the hydroxyapatite. These conditions include a broad temperature range; room temperature is preferred. Flow rate may also vary greatly, and a preferred range is approximately 90 cm/hour.
After the VLPs are bound to the hydroxyapatite, the next step is recovery of the purified VLPs from the hydroxyapatite with an elution buffer. A preferred elution buffer solution contains a phosphate anion, such as a sodium or potassium phosphate solution. Preferred molar ranges are from about 0.05 M to about 1 M, with approximately. 0.2 M being preferred. The pH of the elution buffer should range from about pH 6-8, with a pH of about 7 being preferred.
Other advantages of the method of this invention include:
(a) no requirement for special chromatography equipment and techniques; (b) the method is rapid, requiring no buffer changes; and (c) the method provides an excellent yield of HPV L1.